BME Masters Project: Adrienne Whitney- “Multi-Channel Flow Cytometry Assay Improvement for Pre-Manufacture CAR T-Cell Material Phenotyping”

Master’s Project Defense

Tuesday, April 30th, 2024

Gateway Park, Room GP 1002

1:00pm — 2:00pm

“Multi-Channel Flow Cytometry Assay Improvement for Pre-Manufacture

CAR T-Cell Material Phenotyping”

Adrienne Whitney

Abstract: Chimeric antigen receptor (CAR) T-cell therapies are a form of cell-based gene therapy which engineers a given patient’s T-cells to target and destroy cancerous cells in the body upon reinfusion. Through a rigorous, multi-week process, CAR T-cell manufacture enhances T-cell specificity towards cancer cells, enabling heightened immune response against malignant cells. At Bristol Myers Squibb (BMS), flow cytometry assays are used to phenotype pre-manufacture cellular material to ensure the presence of adequate immune cell populations for CAR T-cell manufacture. However, excess debris and fluorescence issues observed in samples processed per the current flow cytometry assay procedure for pre-manufacture CAR T-cell material phenotyping can render test results unusable. I sought to develop an improved assay procedure which reduces the occurrence of these issues, ultimately saving BMS time and valuable resources. Procedural changes including bulk sample red blood cell lysis prior to cell plating, increased manual mixing upon reagent additions, utilization of Brilliant Stain Buffer in sample processing, and longer antibody staining time were investigated. Increased manual mixing and bulk red blood cell lysis was found to decrease debris present in acquired samples, while longer staining time and increased manual mixing were found to increase median fluorescence intensity (MFI) in most immune cell populations of interest. A final assay procedure was developed incorporating procedural changes which supported the project objectives, and the results of the final assay procedure exhibited statistically significant debris reduction, as well as higher MFIs when compared to the unaltered pre-manufacture CAR T-cell material phenotyping assay procedure. For the sponsor, this ability to produce usable data using the new assay procedure more reliably will save the company time and money as less reagents, consumables, equipment, utilities, and personnel are expended on retests.